Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809) and the triple combination Trikafta®. This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in cystic fibrosis therapeutics.

Supplementary Figure 3. The 14-3-3β/CFTRpS753pS768/CY007424 crystalizes as a tetramer in the asymmetric unit. a Two views of the tetramer consisting of two 14-3-3 dimers (A and B, white cartoon), two copies of the CFTRpS753pS768 peptide (green sticks), and two copies of CY007424 (yellow sticks). b Detailed view of the final 2Fo-Fc electron density map (contoured at 1σ) highlighting the region of the two copies of CY007424 facilitating crystallization between the two dimers of 14-3-3.

General Information
Reagents and solvents were of reagent quality or better and were used as obtained from various commercial suppliers unless otherwise noted. Reaction solvents, such as DMF, DCM, DME and THF, were of DriSolv ® , OmniSolv ® (EMD Millipore, Darmstadt, Germany), or an equivalent synthesis grade quality. Reagent grade solvents were employed for: (i) deprotection, (ii) resin capping reactions and (iii) washing sequences. NMP used for coupling reactions was of analytical grade. DMF was adequately degassed by placing under vacuum for a minimum of 30 min prior to use. Ether refers to diethyl ether. Amino acids, Boc-, Fmoc-and Alloc-protected and side chain-protected derivatives, including those of N-methyl and unnatural amino acids, were obtained from commercial suppliers, Broadband Probe (BBI) probe or a Bruker AVANCE II 700 MHz spectrometer equipped with a cryoprobe (5 mm CPDCH 13C-1H/D) and are referenced internally with respect to the residual proton signals of the indicated deuterated solvent. 13 C NMR spectra also were recorded on the AVANCE II, at 176 MHz. Well-defined coupling patterns for observed resonances are indicated with standard abbreviations (s: singlet, d: doublet, t; triplet, q: quartet, br: broad, dd: doublet of doublets, dq: doublet of quartets, etc.) and coupling constants (J) with the number of protons represented by integration of each resonance denoted by "xH".
High resolution mass spectra (HRMS) for accurate mass measurements were performed on an Agilent Technologies LC-TOF 6224 instrument with either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). Aliquots of 0.2 uL were injected into the mass spectrometer using a 0.5 mL/min flow of 50% MeOH/50% H2O (containing 0.1% formic acid) mixture. The capillary voltage was set at 3000 V and mass spectra were acquired in the range 100-3000 m/z. HPLC analyses were performed on an Agilent 1100 system equipped with a photodiode array (PDA) detector for purity assessment and LC-MSD mass spectral detector for identity confirmation at a flow rate of 2 mL/min using S10 a Zorbax SB-C18 (4.6 mm x 30 mm, 2.5 µm) or a Waters Alliance system running at 2 mL/min with an Xterra MS C18 column (4.6 mm x 50 mm, 3.5 µm) with a Model 996 PDA and a Micromass ZQ or Platform II mass spectrometer.
Data was captured and processed utilizing the instrument software packages except for MS spectra, which were processed with version 4.0 of MassLynx software. The standard gradient employed on both the Agilent and Waters systems using H2O and CH3CN as solvents, each containing 0.1% formic acid, was: 0-0.5 min 5% CH3CN, 0.5-5 min 5->100% CH3CN, 5-7 min 100% CH3CN. UPLC analyses were performed on a Waters Acquity system equipped with PDA and MS detectors at 7 mL/min on an Acquity UPLC BEH C18 column (2.1 mm x 50 mm, 1.7 µm) utilizing a standard gradient also with H2O and CH3CN, each containing 0.1% formic acid: 0-1 min 5% CH3CN, pre-packed cartridges, or plates as appropriate for the compound(s) being purified.
The expressions "concentrated/evaporated/removed under reduced pressure" and "concentrated/ evaporated/removed in vacuo" indicates evaporation of volatile materials utilizing a rotary evaporator under either water aspirator pressure or the stronger vacuum provided by a mechanical oil vacuum pump as appropriate for the solvent being removed or, for multiple samples simultaneously, evaporation of solvents and other volatiles utilizing a centrifugal evaporator system (Genevac HT-24, SP Industries, Warminster, PA, USA). Fmoc-S1 Fmoc-S2 S11 of the Fmoc-protected amino acid (Fmoc-S1, 1 eq) in THF (6-7 mL/mmol) under nitrogen was cooled to 0°C. in an ice-salt bath, then isobutyl chloroformate (IBCF, 1.05 eq) and 4-methylmorpholine (NMM, 1.05 eq) added dropwise simultaneously or successively over approximately 5 min. The mixture was stirred at 0°C. for 30 min, then at room temperature (rt) for another 30 min. The white precipitate that formed was filtered into a round bottom flask through a pre-washed Celite ® pad and rinsed with anhydrous ether. The flask was placed under nitrogen in an icebath, and a mixture of sodium borohydride (1.5 eq) in water added in one shot with the neck of the flask left open.

Standard Procedure for the Preparation of Alcohol Building Blocks
Significant gas evolution was observed and the reaction mixture formed a suspension. More water (20 mL) was added, the ice-bath removed, and the reaction stirred rapidly with monitoring by LC-MS and TLC. After 1 h at rt, LC-MS analysis indicated that the reaction was complete. More water was then added and the organic layer extracted with EtOAc (2x). The combined organic layers were washed sequentially with 1 M citric acid (1x), saturated NaHCO3 (1x), water (1x), brine (1x), and dried over anhydrous MgSO4. The mixture was filtered and the filtrate concentrated under reduced pressure to give the alcohol, Fmoc-S2, in 60-80% yield. The product thus obtained was sufficiently pure to be used without further purification for subsequent reactions.

General Procedure for Oxidation of Alcohol Building Blocks to Aldehydes.
A number of different oxidation reagents were successfully utilized to convert alcohols, generally Fmocprotected, to aldehydes for use in attachment by reductive amination. The products were characterized by 1  The following procedure was employed for the transformation of Fmoc-protected amino alcohols to the corresponding amino aldehyde building block components for use in the reductive amination attachment procedure.
In a round-bottomed flask, the alcohol (1 eq), Fmoc-S3, was dissolved in DCM (4 mL/mmol) and DMSO ( The alcohol, Fmoc-S5 (1 eq) was suspended in DCM (1 mL/mmol) and THF (1 mL/mmol). Manganese dioxide (Strem Chemicals, Newburyport, MA, USA) (1.06 eq) was added and the reaction agitated overnight (o/n) on an orbital shaker at 200 rpm. If the reaction was incomplete, additional quantities of MnO2 were introduced with the mixture agitated for 16 h more after each such addition until starting material was consumed. At that point, the reaction solution was filtered and the residual MnO2 agitated with THF, which was used to wash the material in the filter as well. The MnO2 was washed again with THF and agitated, then passed through the filter. The combined filtrate was dried over anhydrous MgSO4, the solid removed, then the solvent evaporated under reduced pressure to leave the aldehyde, Fmoc-S6, as a solid or syrup. 1 H-NMR and LC-MS analysis were consistent with the expected product and indicated sufficient purity to proceed with use in macrocycle assembly.

General Methods for Synthesis of Libraries of Macrocyclic Compounds
An outline of the general strategy used for the synthesis of the macrocyclic compounds is provided in Supplementary Figure 5. Although a split-pool strategy is employed for efficiency in the library preparation, 8,9,10 each macrocycle is ultimately obtained as a discrete individual compound through the utilization of multiple, small, meshed, polypropylene containers with snap closures (MiniKans) equipped with a radiofrequency (Rf) tag to permit tracking of the syntheses and identification of the particular structure contained therein prior to resin cleavage. 11 The general strategy led to macrocyclic compounds containing three, four or five building blocks (compounds 12, 13, 14, respectively in Supplementary Figure 5) with additional details of the various steps involved in the assembly provided in the succeeding procedures. The Fmoc/tBu protection strategy 12,13 was employed for their preparation. To initiate the process, the first building block (BB1), as its acid, was attached directly to 2'-chlorotrityl chloride polystyrene resin 14 under basic conditions. The assembly of subsequent components then employed one of three reactions after removal of the Fmoc group: amide bond formation, Mitsunobu-Fukuyama reaction 15 (which required additional manipulations to effect), or reductive amination. Depending on the number of BBs desired for the target macrocycle, the route was terminated after 3, 4 or 5 total components were joined together. At that stage, sequential N-terminal Fmoc removal, cleavage from the resin support, macrocyclization via amide bond formation, and final deprotection of the remaining protecting groups were performed. The crude products thus obtained were roughly purified using SPE procedures followed by preparative HPLC with MS monitoring and collection triggering. For each final macrocycle thus isolated, the HPLC purity (UV) was determined and the identity confirmation by MS. Note that it was found that in certain instances, purification prior to removal of the side chain protection was performed, for example, if separation from side products and reagents was observed in the crude HPLC to be more easily achieved than at the fully deprotected stage.
The QUEST Library of novel macrocyclic compounds used for the initial HTS for this investigation was prepared as described in the patent literature, which also provides the structures of thousands of its individual members. 16,17,18 Briefly, its construction followed analogous steps to those just described: (i) synthesis of the individual multifunctional, appropriately protected, building blocks, including elements for interaction at biological targets and fragments for control and definition of conformation, as well as moieties that can perform both functions; (ii) assembly of the building blocks, typically in a sequential manner with cycles of selective deprotection and attachment, utilizing standard chemical transformations such as amide bond formation, Mitsunobu reaction and its variants, nucleophilic substitution reactions, reductive amination, and metal-and organometallic-catalyzed coupling; (iii) optionally, selective removal of one or more side chain protecting groups was performed, either during the building block assembly or after assembly was completed, then the resin-bound molecule further reacted with one or more additional building blocks to extend the structure at the selectively unprotected functional group(s); (iv) once assembly of the linear intermediate precursor was completed, the N-terminal functional group was deprotected, the linear precursor cleaved from the resin, followed by cyclization of the compounds, which can S14 involve one or more steps, to form the macrocyclic structures; and (v) removal of all remaining protecting groups and purification to provide the desired final macrocycles.
Upon isolation and characterization, the library compounds were stored individually in the form thus obtained (solids, syrups, gums) or dissolved in an appropriate solvent, for example DMSO. In solution, the compounds were distributed into an appropriate array format for ease of use in automated screening assays, such as in microplates or on miniaturized chips. After isolation and to ensure the integrity of the compounds was maintained prior to and in-between any testing, libraries were stored at or below -70°C as 10 mM solutions in 100% DMSO. For use, the plates were allowed to warm to ambient temperature and diluted with buffer, first to a working stock solution, then further to reach the appropriate test concentrations for use in HTS or other assays. It should be noted, however, that no inherent instability of these macrocycles has been observed even upon standing at ambient temperature for extended periods of time.

General Methods for Solid Phase Chemistry
For the manipulations described here, polystyrene cross-linked with divinyl benzene (PS-DVB) resins were employed with DMF, DCM and NMP (N-methyl-2-pyrrolidone) as the most common solvents. The volume of the reaction solvent required was generally 3-5 mL per 100 mg resin or approximately 0.04 mL/mg resin. When the term "appropriate amount of solvent" is used in the synthesis methods, it refers to this quantity. Reaction stoichiometry was determined based upon the loading of the starting resin (represents the number of active functional sites, as provided by the supplier, usually as mmol/g). The recommended quantity of solvent roughly amounts to a 0.2 M solution of building blocks at 3-5 eq relative to the initial loading of the resin, except for the initial building block attachment to the resin, for which 2.5 eq was sufficient. Solid phase reactions were conducted in round bottom flasks, solid phase reaction vessels equipped with a fritted filter and stopcock, or Teflon-capped jars. The vessel size was selected so that the solvent/resin mixture only filled ~60% of the container to provide adequate space for the resin to be effectively agitated taking into account these PS-DVB resins swell significantly in organic solvents.
Agitations for solid phase chemistry were performed with an orbital shaker (Thermo Scientific, Forma Models 416 or 430, or New Brunswick) at approximately 200 rpm, unless otherwise specified.
The volume of solvent used for the resin washes was a minimum of the same volume as used for the reaction, although more solvent was generally employed to ensure complete removal of excess reagents and other soluble residual by-products (minimally 0.05 mL/mg resin). Each of the resin washes listed in the procedures was performed for a duration of at least 5 min with agitation (unless otherwise specified) in the specific order listed. The number of washings is denoted by "nx" together with the solvent or solution, where n is an integer. In the case of mixed solvent washing systems, they are listed together and denoted solvent 1/solvent 2. After washing, the expression "dried in the usual manner" and analogous expressions mean that the resin was dried first in a stream of nitrogen or argon S15 for 20 min to 1 h, using argon if there was concern over oxidation of the substrate on the resin, and subsequently under vacuum (oil pump usually) until full dryness is attained [minimum 2 h to overnight (o/n)].

General Procedure for Loading of First Building Block to Resin
The following procedure was followed for adding the first protected building block to 2'-chlorotrityl chloride resin (75-80 mg, 1.11 mmol/g) was loaded into each MiniKan. The resin was pre-washed with DCM (2x), then dried under a nitrogen flow for 2 h. In a suitable reaction vessel, Fmoc-NR1-BB1-CO2H (2.5 eq) was dissolved in DCM (0.04 mL/mg resin) and diisopropylethylamine (DIPEA, 5 eq) added, then the solution agitated briefly and the resin introduced. The reaction was agitated o/n at which point the solvent was removed, washed with DMF (2x), then any remaining reactive sites on the resin capped using two successive treatments with MeOH/DIPEA/DCM (2:1:17). After these treatments and removal of the solvent, the resin was washed sequentially with DCM (1x), iPrOH (1x), DCM (1x), ether (1x), then dried in the usual manner.

Standard Procedure for Monitoring Reaction Progress on the Solid Phase (In-Process Quality Control (QC))
Since direct methods usually employed for monitoring reaction progress (TLC, GC, HPLC) are not applicable for solid phase reactions, it was necessary to perform cleavage of a minimal amount of material from the resin support in order to determine the progress of each transformation, such as described in the following procedure.
Representative duplicate samples in MiniKans specifically for this purpose were included within the library so as not to adversely affect the preparation of any of the target molecules. For solid phase reactions involving amines, the Kaiser (ninhydrin) test was also used to monitor progress. 19

General Procedure for Fmoc Deprotection
In an appropriate vessel, a solution of 20% piperidine (pip) in DMF was prepared, an excess added to the resin and the mixture agitated for 30 min. The reaction solution was removed, then this treatment repeated. After draining of the solvent, the resin was washed sequentially with: DMF (2x), iPrOH (1x), DMF (1x), iPrOH (1x), THF (1x), DCM (1x), ether (1x), then dried in the standard manner.
In order to minimize the potential of diketopiperazine formation when N-alkylated-amino acids were present in the BB1 position, 50% pip/DMF was used for Fmoc-deprotection of BB2 and the procedure further modified as follows: agitation of the deprotection solution with the resin was performed for only 5-7 min, then the solvent S16 drained. DMF was added to rinse the resin, the mixture agitated quickly and the solvent removed. The washing sequence as described above was then executed.

General Procedure for Attachment of Amines to Acids
To an appropriate vessel, DIPEA (7 eq) in NMP was added to the acid-containing building block (3.5 eq), HATU if the acid building block is one known to require repeated treatment for optimal results (i.e. N-alkylated and other sterically hindered amino acids), half of the indicated equivalents was used for each of two treatments.

Triacetoxyborohydride
As the preferred method for attachment of aromatic aldehydes, sodium triacetoxyborohydride was employed in the reductive amination process as follows: 1.2-1.5 eq of the Fmoc-protected aldehyde was dissolved in DCM, the S17 amine-containing resin added, then the mixture agitated for 1.5-3.0 h. To this was introduced NaBH(OAc)3 (5 eq) and the reaction agitated o/n with venting at 0.5 h and 1 h. If in-process QC indicated that free starting amine remained on the resin, additional aldehyde (0.6-0.7 eq) was included as part of the repeat treatment along with the reducing agent. Once the reaction was completed, the solvent was removed, then the resin washed sequentially with DMF (2x), iPrOH (1x), MeOH (3:1, 1x), DCM/MeOH (3:1, 1x), iPrOH (1x), THF (1x), DCM (1x), ether (1x) and dried in the standard manner.

Cyanoborohydride and BAP Treatment
For certain benzylic aldehydes, a sequential Borch and BAP reduction process was found to be beneficial. In the first step, the Fmoc-protected aldehyde (3 eq) in NMP/TMOF (1:1) containing 0.5% glacial acetic acid was added to the resin in an appropriate reaction vessel and agitated for 30 min. To the mixture, NaBH3CN (10 eq) was introduced and the reaction shaken for 10 min, then pressure released and agitation continued o/n. Once in-process QC showed the transformation was complete, solvent was removed and the resin washed sequentially with: DMF (2x), iPrOH (1x), DMF (1x), iPrOH (1x), DCM (2x), ether (1x). If an incomplete reaction was indicated, the solvent was removed and the resin suspended in MeOH/DCM/TMOF (2:1:1). To this was added BAP (2-3 eq) and agitation allowed to With the Nos group in place, the following method was used to alkylate the nitrogen under Fukuyama-Mitsunobu conditions 15 for connection of a hydroxy-containing building block (alcohol or phenol, R-OH) to the activated amine component on resin. This procedure was utilized for preparing N-methyl and other N-alkyl components for which the respective individual building block was not commercially available or otherwise difficult to access. The building block (5 eq) was dissolved in THF, 3 Å molecular sieves (200 mg/MiniKan) added and the mixture agitated for 20-30 min before the activated amine containing resin was introduced. After 1.5 h, the solution was cooled to 0°C and the PPh3-DIAD adduct (5 eq, prepared as described below) added. The reaction was agitated o/n while being allowed to warm to rt. The resin was removed by filtration and washed sequentially with: DMF (1x), S18 i-PrOH (1x), DMF (1x), i-PrOH (1x), THF (1x), DCM (1x), ether (1x) then dried in the usual manner. When methanol was used as the alcohol component to prepare N-methylated derivatives, the molecular sieves were not included and the resin added 5 min after the PPh3-DIAD adduct.
After alkylation, the nosyl group was typically removed using the standard method below, then the next building block added or, if the building block assembly was concluded, the precursor was cleaved from the resin and subjected to macrocyclization. Alternatively, in selected cases, the N-Nos group was maintained and its cleavage delayed until the end of the building block assembly or even until after the macrocyclization, since it provided protection of the backbone amide and served to prevent side reactions at that site. 20

Standard Procedure for Nosyl Deprotection
The N-Nos moiety was removed if further chemistry was performed on that nitrogen atom. To effect this, a

Standard Procedure for the Synthesis of PPh3-DIAD Adduct
This reagent was prepared essentially as previously reported. 21 In a round bottom flask under nitrogen, diisopropyl azodicarboxylate (DIAD, 1 eq) was added dropwise to a solution of triphenylphosphine (PPh3, 1 eq) in THF (0.4 M) at 0°C, then the reaction stirred for 30 min at that temperature. The resulting precipitate was collected on a cooled, medium porosity glass-fritted filter, the solid washed with cold THF (DriSolv grade) to remove any color, then with anhydrous ether. The white powder was dried in vacuo (vacuum pump) and stored under nitrogen in the freezer. It was removed from cold storage shortly before each intended use.

General Procedure for Cleavage from 2'-Chlorotrityl Resin
Resin was liberated from the individual MiniKans and transferred into bottom-filtered reaction tubes compatible with the 48 well format of the Mettler Toledo/Bohdan Miniblocks (now available from SiliCycle). To each tube was added 20% HFIP/DCM (1.5 mL), the block covered and agitated on an orbital shaker (460-500 rpm) for 1-2 h. The solution was drained into 7.5 mL tubes arrayed to collect from each of the 48 wells in the Miniblock (pushed out with nitrogen) and the process repeated with an additional quantity of 20% HFIP/DCM (1.5 mL/tube). The resin was then rinsed with DCM (1 mL, again pushed out with nitrogen) and the solvents in the receiving tubes evaporated in vacuo (Genevac). The crude material was obtained as solids, semi-solids, syrups or gums.

General Procedures for Macrocyclization
Different procedures for cyclization and subsequent SPE processing (using pre-packed or manually packed cartridges and microplates) were employed depending on the nature of the backbone of the macrocycle product as either basic (containing a secondary or tertiary amine) or neutral (lacking such an amine moiety). The deprotection cocktails indicated (2 mL/cpd) were added to the crude cyclized products obtained after the SPE processing and the resulting mixtures agitated for ~2 hr. Once HPLC indicated deprotection had been completed, the volatiles were removed in vacuo (Genevac), 95% DMSO/water (1.5 mL) added to each of the residues and then agitated for 5 h at rt. Analyses were performed on the crude deprotected materials prior to being subjected to preparative HPLC purification using the standard methods below.

Characterization of Macrocyclic Compounds
The following macrocycles are representative of the chemotypes identified as the initial hits from the original HTS of the QUEST Library, as well as the validated hits from the subsequent focused library. These selections include the most active PPI stabilizers found from these investigations.